Comparative genomics reveals signature regions used to develop a robust and sensitive multiplex TaqMan real-time qPCR assay to detect the genus Dickeya and Dickeya dianthicola.

AIMS: Dickeya species are high consequence plant pathogenic bacteria; associated with potato disease outbreaks and subsequent economic losses worldwide. Early, accurate and reliable detection of Dickeya spp. is needed to prevent establishment and further dissemination of this pathogen. Therefore, a multiplex TaqMan qPCR was developed for sensitive detection of Dickeya spp. and specifically, Dickeya dianthicola. METHODS AND RESULTS: A signature genomic region for the genus Dickeya (mglA/mglC) and unique genomic region for D. dianthicola (alcohol dehydrogenase) were identified using a whole genome-based comparative genomics approach. The developed multiplex TaqMan qPCR was validated using extensive inclusivity and exclusivity panels, and naturally/artificially infected samples to confirm broad range detection capability and specificity. Both sensitivity and spiked assays showed a detection limit of 10 fg DNA. CONCLUSION: The developed multiplex assay is sensitive and reliable to detect Dickeya spp. and D. dianthicola with no false positives or false negatives. It was able to detect mixed infection from naturally and artificially infected plant materials. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed assay will serve as a practical tool for screening of propagative material, monitoring the presence and distribution, and quantification of target pathogens in a breeding programme. The assay also has applications in routine diagnostics, biosecurity and microbial forensics.

Phenotypic and genotypic profiling of antimicrobial resistance in enteric Escherichia coli communities isolated from finisher pigs in Australia.

OBJECTIVE: To assess herd-to-herd variation in antimicrobial resistance phenotypes and associated antimicrobial resistance genes (ARGs) in faecal commensal Escherichia coli communities isolated from Australian slaughter-age pigs. METHODS: Hydrophobic grid-membrane filtration (HGMF) was used to screen populations of E. coli isolated from faecal samples obtained from pigs prior to or at slaughter. Multiplex PCRs were applied to the pooled DNA extracted from the samples to identify specific ARGs. METHODS: Pooled faecal samples from 30 finishers, from 72 different Australian pig farms, produced 5003 isolates for screening. HGMF techniques and image analysis were used to confirm E. coli resistance phenotypes to four antimicrobial agents (ampicillin, gentamicin, florfenicol and ceftiofur) using selective agars. Multiplex PCRs were performed on DNA from pooled samples for 35 ARGs associated with seven chemical classes. RESULTS: The prevalence of E. coli isolates showing no resistance to any of the drugs was 50.2% (95% confidence interval (CI) 41.8-58.6%). Ceftiofur resistance was very low (1.8%; CI 0.8-3.9%) and no ARGs associated with 3rd-generation cephalosporin resistance were detected. By contrast, ampicillin (29.4%, CI 22.8-37.0%), florfenicol (24.3%, CI 17.8-32.3%) and gentamicin (CI 17.5%, 10.7-27.2%) resistance prevalence varied greatly between farms and associated ARGs were common. The most common combined resistance phenotype was ampicillin-florfenicol. CONCLUSION: The use of registered antimicrobials in Australian pigs leads to the enteric commensal populations acquiring associated ARGs. However, despite a high intensity of sampling, ARGs imparting resistance to the critically important 3rd-generation cephalosporins were not detected.

Antimicrobial resistance and virulence gene profiles in multi-drug resistant enterotoxigenic Escherichia coli isolated from pigs with post-weaning diarrhoea.

This study aimed to characterize antimicrobial resistance and virulence genes in multi-drug resistant enterotoxigenic Escherichia coli (ETEC) isolates (n=117) collected from porcine post-weaning diarrhoea cases in Australia (1999-2005). Isolates were serotyped, antibiogram-phenotyped for 12 antimicrobial agents and genotyped by PCR for 30 plasmid-mediated antimicrobial resistance genes (ARGs), 22 intestinal and 38 extraintestinal E. coli virulence genes (VGs). Nine serogroups were identified, the most prevalent being O149 (46.2%), O141 (11.2%) and Ont (31.6%). None of the isolates showed resistance to ceftiofur or enrofloxacin and 9.4% were resistant to florfenicol. No corresponding extended-spectrum/AmpC ß-lactamase, fluoroquinolone or floR ARGs were detected. An antimicrobial resistance index (ARI) was calculated from the combined data with a weighting for each antimicrobial agent dependent upon its significance to human health. Serogroup O141 isolates had a significantly higher ARI due to an elevated prevalence of aminoglycoside ARGs and possession of more virulence genes (VGs), including ExPEC or EHEC adhesins (bmaE, sfa/focDE, fimH, ihA) in toxin-producing strains that lacked the normally associated F4 and F18 fimbriae. Few associations between ARGs and VGs were apparent, apart from tetC, sfa/focDE and ompT which, for a sub-set of O141 isolates, suggest possible plasmid acquisition from ExPEC. The multi-drug resistant ETEC ARG/VG profiles indicate a high probability of considerable strain and plasmid diversity, reflecting various selection pressures at the individual farm level rather than emergence and lateral spread of MDR resistant/virulent clones.

Genetic relatedness and virulence gene profiles of Escherichia coli strains isolated from septicaemic and uroseptic patients.

We investigated the relationship between clonality and virulence factors (VFs) of a collection of Escherichia coli strains isolated from septicaemic and uroseptic patients with respect to their origin of translocation. Forty septicaemic and 30 uroseptic strains of E. coli were tested for their phylogenetic groupings, genetic relatedness using randomly amplified polymorphic DNA (RAPD), biochemical fingerprinting method (biochemical phenotypes [BPTs]), adherence to HT-29 cells and the presence of 56 E. coli VF genes. Strains belonging to phylogenetic groups B2 and D constituted 93% of all strains. Fifty-four (77%) strains belonged to two major BPT/RAPD clusters (A and B), with cluster A carrying significantly (P = 0.0099) more uroseptic strains. The degree of adhesion to HT-29 cells of uroseptic strains was significantly (P = 0.0012) greater than that of septicaemic strains. Of the 56 VF genes tested, pap genes was the only group that were found significantly (P < 0.0001) more often among uroseptic isolates. Phylogenetic group B2 contained a significantly higher number of strains carrying pap genes than those in group D. We conclude that uroseptic E. coli are clonally different from septicaemic strains, carry more pap genes and predominantly adhere more to the HT-29 cell model of the gut.